Partial Depletion of Microglia Attenuates Long-Term Potentiation Deficits following Repeated Blast Traumatic Brain Injury in Organotypic Hippocampal Slice Cultures

Abstract

Blast-induced traumatic brain injury (bTBI) has been a health concern in both military and civilian populations due to recent military and geopolitical conflicts. Military service members are frequently exposed to repeated bTBI throughout their training and deployment. Our group has previously reported compounding functional deficits as a result of increased number of blast exposures. In this study, we further characterized the decrease in long-term potentiation (LTP) by varying the blast injury severity and the inter-blast interval between two blast exposures. LTP deficits were attenuated with increasing inter-blast intervals. We also investigated changes in microglial activation; expression of CD68 was increased and expression of CD206 was decreased after multiple blast exposures. Expression of macrophage inflammatory protein (MIP)-1α, interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1, interferon gamma-inducible protein (IP)-10, and regulated on activation, normal T cell expressed and secreted (RANTES) increased, while expression of IL-10 decreased in the acute period after both single and repeated bTBI. By partially depleting microglia prior to injury, LTP deficits after injury were significantly reduced. Treatment with the novel drug, MW-189, prevented LTP deficits when administered immediately following a repeated bTBI and even when administered only for an acute period (24 h) between two blast injuries. These findings could inform the development of therapeutic strategies to treat the neurological deficits of repeated bTBI suggesting that microglia play a major role in functional neuronal deficits and may be a viable therapeutic target to lessen the neurophysiological deficits after bTBI.

Keywords: electrophysiology; hippocampus; long-term potentiation; microglial depletion; repeated blast traumatic brain injury.

FIG. 1.

Long-term potentiation (LTP) following repeated blast-induced traumatic brain injury to organotypic hippocampal slice cultures (OHSCs). (A) LTP was decreased in all injured groups that were tested, but LTP was significantly decreased in OHSCs exposed to two level 3 blasts after both a 1- and 3-day inter-blast interval. (B) Representative electrophysiological traces from an electrode in the CA1 region of a repeated sham-injured OHSC and a repeated level 3 injured OHSC (with a 1-day interval) is included. Mean ± standard error of the mean; n ≥ 8; *p < 0.05 compared with sham.

FIG. 2.

Microglial activation following blast-induced traumatic brain injury (bTBI). Representative immunohistochemical staining of Hoechst (nuclei), (A) CD68, (B) CD206, and (C) Iba1 following a sham or repeated level 3 bTBI (scale bar = 20 μm). (D) CD68 expression was significantly upregulated following repeated injury. (E) CD206 expression was significantly downregulated following repeated injury. (F) There was no change in Iba1 expression following bTBI. Representative bands from Western blots are shown. Mean ± standard error of the mean; n ≥ 3; *p < 0.05, as compared with sham.

FIG. 3.

Microglial depletion following Clodrosome or pexidartinib administration. Representative immunohistochemical staining of Hoechst (nuclei) or Iba1 following either (A) Encapsome (25 μg/mL) or Clodrosome (25 μg/mL) administration or (B) vehicle or pexidartinib administration. (C) Iba1 expression was reduced in organotypic hippocampal slice cultures treated with Clodrosome (10 μg/mL and 25 μg/mL) or pexidartinib (1 μM). Protein expression was evaluated 24 h following 100 ng/mL LPS interrogation. Representative bands from Western blots are shown. Mean ± standard error of the mean; n ≥ 3; *p < 0.05 compared with Encapsome treatment; #p < 0.05 compared with vehicle treatment.

FIG. 4.

Long-term potentiation (LTP) in organotypic hippocampal slice cultures (OHSCs) treated with either vehicle or Clodrosome (25 μg/mL) before and after repeated mild blast-induced traumatic brain injury (bTBI). LTP was significantly decreased after injury in vehicle treated OHSCs. Clodrosome significantly improved LTP following repeated bTBI. Mean ± standard error of the mean; n ≥ 8; *p < 0.05.

FIG. 5.

Effects of microglial depletion on long-term potentiation (LTP) in organotypic hippocampal slice cultures (OHSCs) treated with either vehicle or pexidartinib (1 μM) before and/or after repeated mild blast-induced traumatic brain injury. Pexidartinib treatment of sham OHSCs did not significantly decrease LTP. LTP was significantly improved after blast by pexidartinib treatment before and after injury, only after injury, or only before injury. Mean ± standard error of the mean; n ≥ 7; *p < 0.05, #p < 0.05, §p < 0.05 compared with vehicle treated OHSCs.

FIG. 6.

Effects of MW-189 treatment on long-term potentiation (LTP) in organotypic hippocampal slice cultures following repeated blast-induced traumatic brain injury (bTBI). (A) MW -189 (30 μM) administration immediately following the first of two bTBIs prevented LTP deficits two days after the final injury exposure. LTP also improved when MW-189 was administered only for one day following the first of two repeated bTBIs. (B) Injury and treatment paradigm. Mean ± standard error of the mean; n ≥ 6; *p < 0.05, #p < 0.05 compared with treatment-matched saline vehicle.

FIG. 7.

Changes in cytokine and chemokine expression following a single or repeated blast-induced traumatic brain injury (bTBI). (A-E) Expression was significantly increased for macrophage inflammatory protein (MIP)-1α, interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1, interferon gamma-inducible protein (IP)-10, and regulated on activation, normal T cell expressed and secreted (RANTES) 3 h following injury. However, there were no differences in any of these cytokines at 24 h after either single or repeated bTBI. (F) IL-10 expression significantly decreased 24 h following both single and repeated bTBI. Mean ± standard error of the mean; n ≥ 3; *p < 0.05 compared with time-matched sham; #p < 0.05 compared with time-matched sham.